The first organ system to form in the developing mouse embryo is the cardiovascular system. Defects within the heart and vasculature are largely responsible for embryonic lethality in utero. Within the developing vascular network of the yolk sac as well as in certain intra embryonic regions, endothelial and hematopoietic cells arise in close spatial and temporal association and are thought to derive from a common mesodermal progenitor, the "hemangioblast." Recent work has indicated that hemangioblasts may also have smooth muscle cell potential. Regulation of hemangioblast and embryonic hematopoietic stem cell formation and cell fate specification are still not well understood. This proposal focuses on the characterization and functional analysis of specific subsets of mesodermal cells that give rise to hematopoietic, vascular endothelial, and smooth muscle cells during development. First, we will evaluate the hematopoietic potential and functional activity in vitro and in vivo of prospectively identified mesodermal stem/progenitorcell populations from differentiating embryonic stem cells (embryoid bodies) and from early mouse embryos. Cells will be isolated by flow cytometry on the basis of their expression of primitive cell surface markers and, using a GFP reporter transgene, on the basis of their expression of mMix, a homeodomain transcriptionfactor expressed in embryoid body subsets thought to contain hemangioblasts. The sorted cells will be analyzed using several different assays for stem/progenitor cells in culture and transplantation models in the mouse. Second, in analogous studies, we will evaluate the endothelial and smooth muscle potential and functional activity in vitro and in vivo of prospectively identified mesodermal stem/progenitor cell populations. Third, we will evaluate the role of the Mix homeodomain protein on the developmental potential and functional activity in vitro and in vivo of embryoid body derived mesodermal stem/progenitor cell populations. The origin of embryonic hematopoietic / vascular stem cells and their relationship to stem cells of the adult are unknown. With the increasing focus on regenerative medicine and interest in potential therapeutic applications of human embryonic and adult stem cells, the characterization of mesodermal stem/progenitor cell populations takes on high significance.